The myocyte membrane in the developing heart differentiates new functional properties between the time the primitive tubular myocardium first firms and the stage some two weeks later when its physiological differentiation is virtually complete. These developments include alterations in the mechanism of generation of spontaneous rhythmic action potentials and development of an insulin-sensitive amino-acid transport system. We have demonstrated during the tenure of the present grant period that the spheroidal heart cell aggregate represents a developmentally-defined system that reflects accurately the differentiated properties of the intact donor tissue at each stage, but is more readily amenable to analysis. With the aggregate system, we have: defined the appearance of a fast Na ion conductance mechanism during development using voltage clamp technique, characterized the oscillatory and excitable properties of the membrane in 7-day heart cells, shown that cellular electrical parameters are modified by alterations in the glycoprotein surface coat, and demonstrated that the mode of regulation of the insulin-responsive amino-acid transport system by insulin and amino acids changes between 7 and 14 days of development and that synthesis of insulin receptors may be regulated by the hormone itself. It is the goal of the requested renewal grant to measure the passive electrical parameters and oscillatory properties of the heart cell membrane and to characterize the voltage dependence and magnitudes of the transmembrane currents in aggregates from chick and mouse heart cells at various stages throughout development, in normal and mutant tissue, and in preparations after molecular dissection of surface glycoproteins and in the presence of insulin, ouabain and specific current-blocking agents. Similar preparations will be employed for measurements of amino acid transport under basal and insulin-stimulated conditions, specific hormone receptors, 42K-influx, insulin-induced hyperpolarization and 3H-ouabain binding. These measurements are designed to elucidate the mechanisms of differentiation of the functional properties of the membrane, and the inter-relations between the electrical parameters and amino-acid transport systems in the heart cell.